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IL-11 has been detected in inflamed joints; however, its role in the pathogenesis of arthritis is not yet clear. Studies were conducted to characterize the expression and functional significance of IL-11 and IL-11Rα in rheumatoid arthritis (RA). IL-11 levels were elevated in RA synovial fluid (SF) compared to osteoarthritis (OA) SF and plasma from RA, OA and normal individuals (NLs). Morphologic studies established that IL-11 was detected in lining fibroblasts and macrophages in addition to sublining endothelial cells and macrophages at higher levels in RA compared to NL synovial tissues. Since IL-11Rα was exclusively expressed in RA fibroblasts and endothelial cells, macrophages were not involved in IL-11 effector function. Ligation of IL-11 to IL-11Rα strongly provoked fibroblast infiltration into RA joint, while cell proliferation was unaffected by this process. Secretion of IL-8 and VEGF from IL-11 activated RA fibroblasts was responsible for the indirect effect of IL-11 on endothelial cell transmigration and tube formation. Moreover, IL-11 blockade impaired RA SF capacity to elicit endothelial cell transmigration and tube formation. We conclude that IL-11 binding to endothelial IL-11Rα can directly induce RA angiogenesis. In addition, secretion of proangiogenic factors from migrating fibroblasts potentiated by IL-11 can indirectly contribute to RA neovascularization.

BackgroundIn patients with rheumatoid arthritis (RA) different joints were shown to share the same dominant T-cell clones, suggesting shared characteristics of the inflammatory process and indicating that strategies to selectively target the antigen receptor might be feasible. Since T- and B-lymphocytes closely interact in adaptive responses, we analysed to what extent different joints also share dominant B-cell clones.MethodsIn 11 RA patients, quantitative B-cell receptor (BCR) repertoire analysis was performed in simultaneously obtained samples from inflamed synovial tissue (ST) from distinct locations within one joint, from multiple joints, from synovial fluid (SF) and peripheral blood (PB).ResultsST biopsies from different locations in the same joint showed clear overlap in the top-25 dominant BCR clones (16.7%, SD 12.5), in the same range as the overlap between ST and SF in the same joint (8.0%, SD 8.8) and the overlap between ST-ST between different joints (9.1%, SD 8.2), but clearly higher than the overlap between ST and PB (1.7%, SD 2.4; p<0.05) and SF and PB (2.7%, SD 4.1; p<0.05). Interestingly, these figures were substantially lower than the overlap observed in previous T-cell clonality studies.ConclusionsWe conclude that in RA BCR clonal responses may be more localized than TCR clonal responses, pointing to antigen-selective influx, proliferation and/or maturation of B-cells. B lineage cells in the SF may adequately represent the dominant BCR clones of the ST, which is in contrast to T-cells. Collectively, the presence of shared B- and especially T-cells in different joints from the same patient suggests that approaches might be feasible that aim to develop antigen-receptor specific targeting of lymphocyte clones in RA as an alternative to more generalized immunosuppressive strategies.

Background. Synovial collagen remodelling may mirror chronic inflammation and repair processes, potentially serving as a structural correlate of disease activity in arthritis. Based on this, the aim of this study was to investigate the relationship between synovial inflammatory histopathology and collagen remodelling in patients with active rheumatoid arthritis (RA) or psoriatic arthritis (PsA).   Methods. US-guided synovial biopsies were obtained from inflamed joints (knee/wrist) in 30 patients (18 RA, 12 PsA) with active arthritis, undergoing therapeutic re-evaluation.Synovial inflammation was evaluated considering Krenn Synovitis Score (KSS), immunohistochemical analysis (sublining CD68, CD3, CD20, CD138), pathotype classification, and evaluation of neutrophil infiltration.For 12 patients (RA, PsA),3-micrometer sections were stained with fresh Picrosirius red,then digitized via brightfield and polarized light microscopy for morphometry and collagen typing.Stained area and collagen maturation index were quantified using computer-aided image analysis.Clinical disease activity was recorded at baseline and at 3-month follow-up.   Results. The mean age of patients was 52 years and 64.5% were female;the mean disease duration was 5.6 years. Mean KSS was 4.5±0.5 in RA and 4.2±0.5 in PsA. 17 (56.7%) patients were classified as lympho-myeloid, 10(33.3%) as diffuse-myeloid,and 3(10%) as pauci-immune pathotype.Moreover, neutrophil infiltration of ST was present in 12(40%) of the overall cohort and was associated with higher KSS (p = 0.003).Higher KSS also tended to correlate with the number of previous bDMARDs, and pathotype distribution differed by prior biologic exposure (p = 0.029);notably, patients with a pauci-immune synovitis had received the highest number of previous bDMARDs. Considering collagen deposition,the mean collagen area was 39.48±7.52% in RA and 40.79±12.09% in PsA, while the mean collagen maturity index was 0.73±0.04 and 0.72±0.03, respectively.No associations were found between collagen parameters and sex, age, disease duration, or biopsy site. In RA, collagen area showed a trend toward inverse correlation with CD68+ macrophage infiltration (p = 0.057). In PsA, both collagen area and maturation index were inversely correlated with inflammatory infiltrate, B cells, and plasma cells (all p = 0.04). Additionally, disease duration in PsA was positively associated with both collagen area(p = 0.025) and collagen maturity index (p = 0.017), suggesting that long-standing disease may promote extracellular matrix stabilization. Importantly, a significant inverse correlation between collagen area and collagen maturity index was observed in RA (p = 0.047). At 3-month follow-up, histological pathotype was associated with DAS28 response: responders more frequently showed a diffuse-myeloid profile, while no responders were mostly observed among pauci-immune cases.   Conclusions. Synovial immune cell infiltration is associated with collagen remodelling in both RA and PsA, with distinct patterns linked to disease duration and tissue organization.The inverse correlation between collagen area and maturity in RA suggests matrix expansion during active synovitis.

Synovial tissue is a key structure in diarthrodial joints and is the primary target of inflammation in autoimmune arthritis. The study of synovial tissue has developed significantly in the last two decades as arthroscopic and ultrasonographic techniques have allowed visualization and access to synovial biopsy. Further progress in synovial tissue processing and analysis has improved studies of disease pathogenesis, biomarker discovery, and molecular therapeutic targeting with increasingly specialized analytical and technological approaches. In September 2018 the first course on Synovial Tissue Biopsies was convened in Brussels, in this Mini Review these approaches will be described and I will summarize how synovial tissue research advanced.

Background. Patients meeting the EULAR difficult-to-treat rheumatoid arthritis (D2TRA) definition are classically non-responders to TNF inhibitors (TNFi). As such, they can be treated with biological drugs with other mechanisms of action (OMA; tocilizumab, sarilumab, abatacept, rituximab) or Janus Kinase inhibitors (JAKi). The aim of the study was to investigate whether characteristics of synovial tissue may predict treatment response to OMA or JAKi in D2TRA patients.   Materials and Methods: The MATRIX-D2T was a prospective cohort, multi-centre study that included RA patients with moderate to high disease activity undergoing ultrasound-guided synovial tissue biopsy (UGSTB) at two institutions. In this post-hoc analysis, patients were eligible for inclusion if they fulfilled the EULAR D2TRA definition. The primary outcome was a change in CDAI after six months; the secondary outcome was low disease activity (LDA or CDAI<=10). Predictors were synovial grading according to Krenn Synovial Score (2-4, low grade; 5-9, high grade); synovial pathotypes (lympho-myeloid, diffuse-myeloid, pauci-immune fibroid); and the count of lymphoid aggregates. Outcome comparisons between treatment groups (JAKi vs. OMA) were assessed through repeated measures ANOVA corrected for ACPA titre.   Results. Among 132 patients undergoing USGTB during the study period, 26 patients met the study criteria and were included (mean (SD) age 58 (11) years, duration 15 (10) years, females 62%). Twelve patients initiated JAKi and 14 OMA; the two groups were comparable for baseline characteristics (Table 1). The median (IQR) number of failed b/tsDMARDs was 3 (3), and for csDMARDs, it was 2 (3). Median (IQR) KSS was 5 (7). After 6 months, the mean (SD) CDAI significantly decreased more in JAKi than OMA (mean difference -10.7, 95% CI -19.4 to -2.0, p=0.018). Compared to OMA, a stronger CDAI decrease with JAKi was shown in patients having high-grade synovitis only (Figure 1a-b), with no clear association with synovial pathotypes or the presence of lymphoid aggregates. Patients in LDA were numerically more frequent in JAKi recipients (n=6/12, 50%) compared to OMA (n=3/14, 21.4%; p=0.218; Figure 1c).   Conclusions. Our results suggest that UGSTB may help predict clinical response to JAKi or OMA in DT2RA patients. However, the sample size was small, and results must be confirmed in larger independent cohorts.  

Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4 and CD8 T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

We investigated the potential involvement of pyroptosis, a proinflammatory form of regulated cell death, in rheumatoid arthritis (RA). Synovial fluid, synovial tissues and/or serum were compared among 32 patients with RA, 46 patients with osteoarthritis (OA) and 30 healthy controls. Samples were assayed for interleukin (IL)‐1β, IL‐18 and lactate hydrogenase (LDH). Synovial expression of NLRP3, caspase‐1 and cleaved gasdermin D (GSDMD) was assayed using immunohistochemistry and multiplex immunohistochemistry. Patients with RA showed significantly higher levels of IL‐1β and IL‐18 in synovial fluid than patients with OA, and significantly higher levels of both cytokines in serum than healthy controls. RA was associated with higher levels of LDH in synovial fluid than OA. Among patients with RA, levels of IL‐1β, IL‐18 and LDH were significantly higher in synovial fluid than in serum, and the levels in synovial fluid positively correlated with disease activity and inflammation. Synovial cells, particularly macrophages, showed upregulation of NLRP3, caspase‐1 and cleaved GSDMD in RA compared to OA. Our results implicate pyroptosis in the pathogenesis of RA, perhaps as a driver of local inflammation in joints.

Effective treatment of osteoarthritis (OA) remains a huge clinical challenge despite major research efforts. Different tissues and cell-types within the joint contribute to disease pathogenesis, and there is great heterogeneity between patients in terms of clinical features, genetic characteristics and responses to treatment. Inflammation and the most abundant immune cell type within the joint, macrophages, have now been recognised as possible players in disease development and progression. Here we discuss recent findings on the involvement of synovial inflammation and particularly the role of synovial macrophages in OA pathogenesis. Understanding macrophage involvement may hold the key for improved OA treatments.